The present invention relates generally to methods and compositions for treating plants and for plant growth enhancement.
Photosynthesis is the process by which all photosynthetic plants utilize solar energy to build carbohydrates and other organic molecules from carbon dioxide (CO.sub.2) and water. In general, photosynthesis is a complex sequence of electron and proton-transfer reactions leading to stable reduced metabolites; but when electrons or radicals accumulate along this chain, the resulting imbalance interferes with one after another system until growth decreases. The chemistry of living systems dictates that an electron acceptor is usually balanced by the presence of an electron donor, however, application of chemicals for biological response has generally been one-sided in the sense that xenobiotics are generally formulated without regard to balancing electron acceptors and donors. This historical one-sided approach often stresses the biological system when either oxidants or reductants abound. Our tests suggest that when a balance of electron couples is established by application of formulations selected for appropriate pairing, stress components may be neutralized. The E.sub.0 values of the prospective couples are defined within a range that is compatible to biological systems. In sunlight, a nontoxic balance is especially important for minimizing damage by oxidants. Imbalances of electron couples may be corrected by induction of Cytochromes P450 (CYP) and NADPH:Cytochrome P450 reductase (CPR) pathways that result in the utilization of reducing power.
Cytochromes P450 are a superfamily of hemoproteins that catalyze the singular insertions of oxygen, i.e. monooxygenation, of endogenous and xenobiotic hydrophobic substrates, wherein, the general reaction for hydroxylation by the cytochromes P450 system is, EQU RH+NADPH+H.sup.+ +O.sub.2 .fwdarw.ROH+NADP.sup.+ +Water,
and R represents a substrate compound. The CYP and flavin monooxygenase families are noted for their broad substrate specificities and utilization of oxygen without being linked to phosphorylation of adenosine diphosphate (ADP) and can mediate hydroxylations at nitrogen and sulfur heteroatoms, epoxidations, dehalogenations, deaminations and dealkylations. In general, the monooxygenations require one or two additional proteins to transfer electrons from NADPH to the heme iron and these systems are placed in two groups: Class I, which use an iron-sulfur protein to shuttle electrons from FAD-containing reductase to CYP in mitochondria and bacteria; and Class II, in which NADPH:Cytochrome P450 reductase transfers electrons from NADPH to a CYP in microsomes. In plants, CYP comprises a wide range of hydroxylases, epoxidases, peroxidases and oxygenases which are largely based upon Class II monooxygenations.
Neither the direct connection of CYP and CPR to regulate photosynthesis nor the formulations of cytochromes P450 and inducer substrates have been made previously. We introduce novel methods for formulating compositions comprised of CYP and CPR substrates and enzymes selected for completing the necessary electron couples and inducing the enzymes. These formulations enhance plant growth, improve activity and prevent phytotoxicity.
For these reasons, it would be desirable to provide novel methods and formulations for activating cytochromes P450 enzymes. It would be particularly desirable if such methods and compositions were able to regulate plant growth (i.e., PGR). Additionally, it would be desirable if the compositions reduce toxicity of otherwise one-sided treatments. The present invention should further provide convenient methods resulting in increased activities of CYP and CPR electron couples for applying the novel compositions to plants. It is desirable that the methods and compositions of the present invention promote rapid growth and maturing of the treated plant, increase sugar content, improve blossoms and enhance the quality and quantity of plants. Furthermore, it is generally desirable to provide methods for enhancement of CYP and CPR related enzymes in all biological systems.
The structures and functions of CPR and CYP are reviewed with focus on animal CYP, some of which metabolize more than fifty structurally diverse compounds. See, e.g., H. W. Strobel, et al., "NADPH Cytochrome P450 Reductase and Its Structural and Functional Domains," and C. von Wachenfeldt, et al., "Structures of Eukaryotic Cytochrome P450 Enzymes," P. R. Ortiz de Montellano, ed. (1995) CYTOCHROME P450: STRUCTURE, MECHANISM, AND BIOCHEMISTRY (Second Edition), Plenum Press, New York. Inducers of cytochromes P450 in animal systems include aromatic hydrocarbons, proteins, phenobarbital, peroxisome proliferators, steroids, aminopyrine and ethanol (see, J. P. Whitlock et al., "Induction of Cytochrome P450 Enzymes That Metabolize Xenobiotics" in P. R. Ortiz de Montellano, ed. (1995) CYTOCHROME P450: STRUCTURE, MECHANISM, AND BIOCHEMISTRY (Second Edition), Plenum Press, New York, pp 367-390); but no inducers of cytochromes P450 for growth have been identified in green plant systems.
In avocado tissue, alcohols, aniline, p-chloro-N-methylaniline, N, N-dimethylaniline, cinnamic acid, dimethyl formamide, aryl hydrocarbons and fatty acids showed binding to cytochromes P450. See, S. Cottrell, et al., "Studies on the cytochrome P-450 of avocado (Persa americana) mesocarp microsomal fraction" Xenobiotica 20: 711-726 (1990). In recent reviews of molecular cloning, plant pathways included cytochromes P450 catalysis of oxygen insertion for fatty acids, phenylpropanoids, flavonoids, terpenoids, alkaloids, dyes, pesticides (see, e.g., G. P. Bolwell, et al., "Review Article Number 96. Plant Cytochrome P450" Phytochemistry 37: 1491-1506 (1994)); lignins, coumarins, pigments, alkaloids, jasmonates and plant growth regulators (see, M. A. Schuler "Plant Cytochrome P450 Monooxygenases" Critical Reviews in Plant Sciences 15(3): 235-284 (1996)). Metolachlor is a herbicide that is detoxified by cytochromes P450 (see, D. E. Moreland, et al., "Metabolism of Metolachlor by a Microsomal Fraction Isolated from Grain Sorghum (Sorghum bicolor) Shoots" Z. Naturforsch 45c: 558 (1990)). Beneficial effects of flower inducement implicate binding of carbamates to cytochromes P450 (see, M. Kusukawa, et al., "N-(3,4-Methylenedioxyphenyl)carbamates as Potent Flower-Inducing Compounds in Asparagus Seedlings as Well as Probes for Binding to Cytochrome P-450" Z. Naturforsch 50c: 373 (1995)), where known inhibitors of cytochromes P450 including piperonyl butoxide and trans-cinnamic acid 4-hydroxylase stopped the effect. The hormonal action of the ecdysone-like brassinosteroids that regulate various aspects of plant development is related to CYP90 genes (see, M. Szekeres, et al., "Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450 controlling cell elongation and de-etiolation in Arabidopsis" Cell (Cambridge) 85: 171 (1996)). Salicylate and aspirin caused elevation of rat liver ethanol inducible cytochromes P450 (see, B. Damme, et al., "Induction of hepatic cytochrome P4502E1 in rats by acetylsalicylic acid or sodium salicylate" Toxicology 106: 99-103 (1996)) and, although salicylates in plants are associated with systemic acquired resistance, their relationships to plant cytochromes P450 has not been demonstrated (see, e.g., S. A. Bowling, et al., "A Mutation in Arabidopsis That Leads to Constitutive Expression of Systemic Acquired Resistance" The Plant Cell 6: 1845-1857 (1994)). Phenobarbital has been shown to enhance the activity of CYP.sub.cc in non-photosynthetic plant tissue cultures. See, J. Palazon, et al., "Effects of auxin and phenobarbital on morphogenesis and production of digitoxin in Digitalis callus" Plant and Cell Physiology 36: 247 (1995).
As a supplement to tissue culture, tyrosine has been found in specific natural products during fermentation. See, Y. Hara, et al., "Effect of gibberellic acid on berberine and tyrosine accumulation in Coptisjaponica" Phytochemistry 36: 643-646 (1994)). Tyrosine is essential for flavin mononucleotide binding to cytochromes P450, (see, M. L. Klein, et al., "Critical Residues Involved in FMN Binding and Catalytic Activity in Cytochrome P450.sub.BM-3 " The Journal of Biochemistry 268: 7553-7561 (1993)) and plays a key role in facilitating electron transfer between flavin mononucleotide and heme groups of other cytochromes (see, C. S. Miles, et al., "Tyr-143 facilitates interdomain electron transfer in flavocytochrome b.sub.2 " Journal of Biochemistry 285: 187-192 (1992)). Tyrosine is a substrate for CYP56 and CYP79 in plants. See, B. M. Koch, et al., "The primary sequence of cytochrome P450tyr, the multifunctional N-hydroxylase catalyzing the conversion of L-tyrosine of p-hydroxyphenylacetaldehyde oxime in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench." Archives of Biochemistry and Biophysics 323: 177-186 (1995).
Among the early cytochromes P450 functional markers, para-nitrobenzoate (pNBA) was used to screen the activity of liver microsomal cytochromes P450 substrates by following the reduction to the primary amine. Thus, cytochrome P450 substrates were defined by type I spectra characterized by a trough at 420 nm and a peak at 385 nm or type II spectra characterized by a trough at 390 nm and a peak at 430 nm. See, H. A. Sasame, et al., "Studies on the Relationship between the Effects of Various Substances on Absorption Spectrum of Cytochrome P-450 and the Reduction of p-Nitrobenzoate by Mouse Liver Microsomes" Mol. Pharmacol. 5: 123 (1969); and J. R. Gillette "Reductive Enzymes" Handbuch der experimentellen Pharmakologie 28/2: 349 (1971). In addition to pNBA, other oxidants have been identified including menadione, Mitomycin C, Adriamycin, anthraquinone sulfonate, dinitrobenzene, and quinones, their association with cytochromes P450 dependent upon E.sub.0 values residing within a range of -400 mV to -165 mV. See, J. Butler, et al., "The one-electron reduction potential of several substrates can be related to their reduction rates by cytochrome P-450 reductase" Biochimica et Biophysica Acta 1161: 73 (1993). A review of chemical potentials for electron couples detailing one-electron processes for reduction of oxidants (reduction of electron acceptor) and oxidation of reductants (oxidation of an electron donor) gives values for approximately 700 compounds (see, P. Wardman "Reduction Potentials of One-Electron Couples Involving Free Radicals in Aqueous Solution" J. Phys. Chem. Ref Data 18(4): 1637-1755 (1989) including flavin, bipyridinium, nitroaryl, phenol, terpenoid, imidazole, amine, peroxide and indole compounds. Iodosobenzene and N-oxide of p-cyano-N,N-dimethylalanine have been used for oxidation reactions with CYP in chemical models (see, W. Nee, et al., "Use of N-oxide of p-Cyano-N,N-dimethylalanine as an "Oxygen" Donor in a Cytochrome P-450 Model System" J Am. Chem. Soc. 104: 6123 (1982)), but they have not been applied to plants or other biological systems.
U.S. Pat. No. 5,532,204 proposes foliar applied methanol at the R5 seed growth stage of legumes. U.S. Pat. No. 5,300,540 proposes preservation of freeze-dried plant cells with barrier compositions containing, polyethylene glycol, p-aminobenzoic acid, acetylsalicylic acid, cinnamic acid, benzoic acid, blended alcohol and other organics. U.S. Pat. No. 3,897,241 proposes application of ethanolamine formulations with carboxylic acids of less than 8 carbons, such as, oxalic acid, formic acid, acetic acid, phthalic acid and glutaric acid to fruit-bearing plants. U. S. Pat. No. 4,799,953, proposes polymeric condensates of the sulfur-polymers of thiolactic and thioglycolic acids, increasing the rate of growth and production of chlorophyll specific to tissue and hydroponic culture of Lemna minor. European Patent 465 907 A1 proposes compositions for stimulating the growth and ripening of plants comprised of at least one adduct of menadione bisulfite and a compound chosen from a group including pABA, nicotinamide, nicotinic acid, thiamine, tryptophan, histidine, or adenine. U.K. Patent Application 2 004 856 proposes plant growth stimulating compositions consisting of cysteine as the active component in formulations that also include sulfosalicylic acid, folic acid, an aldehyde, a magnesium salt, and a buffer. European Patent FR 2 689 905 A1 proposes a method for cloning DNA sequences coding for an NADPH Cytochrome P450 reductase implicated by survival of a deficient mutant of Saccharomyces cerevisiae.
PCT WO94/00009 is the published text of parent application PCT/US93/05673 (published on Jan. 6, 1994) and U.S. Pat. No. 5,597,400 issued on Jan. 28, 1997. South African patent 93/4341, which is also the equivalent of PCT/US93/05673, issued on March 30, 1994. South African patent 96/1637, which is also the equivalent U.S. application Ser. No. 08/610,928, filed on Mar. 5, 1996, which was filed as a PCT International Application PCT/US96/02444, on Feb. 20, 1996.